Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives

Definitions

• RNA should be understood to mean ribonucleic acid.
• RNA derivatives refers to ribonucleosides and ribonucleotides as well as compounds which are deriva tives or homologues of these sub-units of RNA.
• a ribonucleoside is an N-glycoside of a heterocyclic base, generally a pyrimidine or purine.
• a ribonucleotide is a phosphoric acid ester of a ribonucleoside and may be ribonucleoside monophosphate or a ribonucleoside polyphoshate.
• RNA polymers found primarily in the cytoplasmic portions of cells, in various degrees of polymerization, are connected with the synthesis of specific proteins required by the cells.
• Monomeric nucleotides of RNA are found within cells in combination especially with many of the B vitamins, in which form they function as coenzymes promoting specific reactions necessary for the normal function of the organism.
• R may be a purine or pyrimidine base, most commonly in nature; adenine, guanine, uracil, or cytosine.
• the vertical heavy line represents a five-carbon sugar in pyranose configuration (d-ribose) and the numbers 3 and 5 represent respectively the third and fifth carbon of this sugar.
• P is a phosphate ester bridge connecting adjacent monomers through a linkage between the 3 and 5' carbons.
• the polymer schematically shown may be degraded in many ways by a variety of enzymes which have been isolated from mammalian tissue, snake venom, and other living cells such as micro-organisms, at an extremely high cost.
• the procedure is thought to be usually stepwise with certain alternative pathways.
• the enzyme with activity responsible for reducing the size of N is called a nuclease or depolymerase. Once this shortening is acice complished the phosphate bridges can be attacked specifically either at points indicated by the dotted lines, or at the wavy lines.
• RNA degradation products Many of the degradation products outlined above have become significant articles of commerce because of their importance in research studies on the chemistry and properties of RNA. .They are also used in pharmaceutical preparations. Also, the 5-ribonucleotides (particularly inosine 5-monoph0sphate (IMP) and guanosine 5-monophosphate (GMP)) have become important as food flavoring enhancers.
• IMP inosine 5-monoph0sphate
• GMP guanosine 5-monophosphate
• the rapidly proliferating parts (the rootlets and stems) of germinating seeds surprisingly constitute a particularly rich source of enzymes which are capable of readily splitting RNA.
• enzymes may be simply and efliciently utilized for such splitting of RNA, particularly for the production of 5-nucleotides.
• the proliferating rootlets of the seeds are the richest source of enzymic material, the stems containing less, but useful quantities, whereas the seed or kernel itself does not contain a commercially attractive quantity.
• the property of the proliferating parts of the seed as a rich source of enzymic material is general property of seeds as a class and is particularly true of monocotyledon seeds.
• Preferable seeds are: those capable of being malted, such as oats, barley, wheat, corn, rye, mullet, sorghums, and
• bean seeds are germinated for the production of bean sprouts, making available a cheap and plentiful supply of these rootlets for the practice of the invention.
• malt sprouts commercially known as malt sprouts
• any readily available seed rootlet or sprout can be substituted, such as, for example, those from wheat malt, rice malt, or rye malt, or bean shoots, or any of those mentioned above.
• the uncomminuted rapidly proliferating parts of germinating seeds are initially treated to obtain an enzymic medium which is capable of hydrolyzing RNA to provide the desired high yields of nucleotides.
• the seed-parts which are normally dry, are first either: soaked in water and then washed with a further quantity of water; or subjected to a multiplicity of washings with water. Where the initial treatment of seed-parts comprises soaking followed by washing, the soaking period should extend for from about 30 to 90 minutes to precondition and hydrate the seedparts. The soaked seed-parts are thereafter washed with water to remove bacteria.
• the washing treatment is particularly important where the seed-parts comprise malted rootlets since such rootlets contain bacteria (resulting from the malting process) which, if not removed, proliterate enzymes destructive to 5-nucleotides.
• the washing may be continuous with the waste water continuously removed or may be accomplished as a series of batch washings with the waste water removed after each wash period.
• the water utilized for soaking the seed-parts and/or washing the seed-parts should not be extremely hard, i.e., contain more than about 1000 parts per million of hardness. Where the natural sources of water yield only hard water, the final washing of seed-parts should be preferably carried out using deionized or soft" water.
• the soaking stage may be eliminated provided the washing period is sufficient to accomplish the desired hydration and preconditioning action.
• small quantities of a suitable bactericide may be added to the wash water utilized during one or more of thewashings.
• seed-parts After suflicient washing, additional water is mixed with the washed seed-parts in a preferred ratio of about 5 to 15 parts by weight of water to one part by weight of solids (seed-parts). Where the seed-parts comprise malt sprouts, the ratio of water to solids may be preferably about to 1. a
• the water/seed-part suspension or slurry is next sub jected to a relatively short heat treatment (about 0.5 to about 7 minutes and preferably 2 to 5 minutes) during which such suspension is agitated.
• a relatively short heat treatment about 0.5 to about 7 minutes and preferably 2 to 5 minutes
• steam may be directly introduced into the suspension in sufficient amount and for a sufficient time to rapidly establish and maintain the temperature of the suspension at between 70 to 85 C. and preferably 70 to 75 C. with optimum commercial results being achieved at about 72 C.
• phosphatase deactivators or inhibitors Prior to the heating period, and while maintaining agitation of the suspension, phosphatase deactivators or inhibitors are added to the solution.
• Such deactivators may comprise: metallic ions (zinc), preferably ZnAc -H O, as a deactivator of 5-ribonucleotidase; and borate ions (such as H BO which may act as a deactivator of general phosphatases.
• metallic ions preferably ZnAc -H O
• borate ions such as H BO which may act as a deactivator of general phosphatases.
• Enzymic media prepared as described heretofore, have a broad spectrum of activity with respect to ribonucleic acid but, in accordance with the further embodiments of this invention, it has been discovered that by careful control of such activity a surprisingly high yield of 5'- nucleotides may be obtained by hydrolyzing such acid with such media in a relatively short time period.
• 5'-nucleotides may be selectively obtained from RNA by the reaction of a digestion solution of RNA and enzyme material heated to from about 60 to 70 C. for from about 1% to about 5 /2 hours.
• the initial pH of the RNA solution may be as high as 8.5 and preferably the pH of the digestion solution does not decrease during hydrolysis to below about 5.2.
• RNA hydrolysis it is important that the seed-parts remain substantially in particulate form during the heat treatment stage and during the subsequent RNA hydrolysis stage.
• a heat treated enzyme medium comprised of water and the rapidly proliferating parts of germinating seeds wherein the seed-parts are not comminuted and remain as particulate matter in the medium.
• Improved enzymic medium to RNA solution ratios are possible. Radically shorter hydrolysis times are required.
• Significantly simplified solids separation procedures are required and there are less non-enzymatic impurities extracted into the hydrolyzate mixture.
• overall processing of the enzymic medium and hydrolyzate mixture is possible together with the attainment of exceptionally high yields of 5'-mononucleotides in the final hydrolyzed solution.
• a ribonucleic acid solution is introduced to a hydrolysis stage together with the enzymic medium (includes seed-parts) from the heat treating stage to form a digestion or hydrolysis solution including suspended seed-parts.
• the nucleic acid solution may comprise RNA, water, and appropriate amount of an alkaline material for adjusting the pH of such solution to the preferred alkaline range. Any of the well-known alkaline pH adjusting materials may be used such as: ammonium hydroxide and ammonium carbonate; sodium hydroxide; tris (hydroxy methyl) amino methane; and primary, secondary and tertiary amines which are water miscible.
• the digestion or hydrolysis solution (enzymic medium including seedparts-l-RNA solution including pH adjustment agent) a may comprise about 24% RNA with the ratio of enzymic medium to RNA solution being from about 1:1 to about 3:1.
• the final digest solution which may have its pH range adjusted periodically (if necessary) to maintain the same within the range of 5.2 to 8.5, is heated to from about 60 to about 70 C. (preferably 63 to 67 tides.
• RNA .--150 liters of RNA solution (30 kg. of commercial RNA and 500 g. NaF) at pH 7.0-7.1 (adjusted with NaOH) and at 20 C. was added rapidly to the above pretreated water/seed-part suspension with the total mixture agitated and the temperature adjusted to about 65 C. Agitation was continued and the temperature maintained at between 64.5" C. and 65.5 C. for 2 hours. Analysis of the hydrolysis solution indicated nucleic :acid degradation to be 95% with 90% 5'-nucleotides present. The hydrolysis was stopped by lowering the temperature of the mixture to 35 C. and adjustiiig the pH to 3.43.6 by the addition of HCl. was filtered to remove the spent plant parts with the filtrate further treated to obtain the desired 5'-nucleotides free of impurities.
• EXAMPLE 11 A further series of experiments was carried out to determine additional processing conditions.
• Experiments p throungh set forth in table on next page, relate to processing wherein uniform lots of dried seed-parts (same malt sprouts and quantities as in Example 10) were: (1) subject to five washings each with water; (2) mixed with water in a ratio of 1.0 kg. of sprouts to 10.0 liters of water; and (3) heat treated as indicated in the table.
• the resulting enzyrnic medium (including malt sprouts) in each instance was then: (1) mixed with a 9.0% RNA solution in a ratio of 2.2 kg. of dried sprouts to 1.0 kg. of commercial RNA (about 90%); and (2) hydrolyzed under varying temperatures and RNA solu- TABLE V Heat Treatment Hydrolyzate Analysis Experi- Hydrolysis,
• Mung Bean Sprouts 1 1 was ground in a Waring Blendor. Both parts were mixed with equal proportions of water with zinc ion added and heat treated at 72 C. for five minutes. The quantities of water and seed-parts were the same as used during the foregoing experiments involving malt sprouts. Both enzymic media (ground grain and unground grains) were mixed with RNA solution at pH 7.0. The final RNA concentration of both mixtures was 3%. NaF was added 2.
• the aqueous enzymic medium claimed in claim 1 in which the rapidly proliferating seed-parts are derived from seeds selected from the group consisting of oats, barley, Wheat, corn, rye, rice, grass, bean, pea, melon, papaya, locust bean, sunflower and sesame seeds.
• impurities which may hamper subsequent separation of the 5'- nucleotides are removed.
• impurities are mainly (I) inorganic phosphates, (2) protein from the enzymic medium, (3) small amounts of unhydrolyzed nucleic acid, and (4) nucleosides.
• the first three can be removed by at least two methods.
• One method involves the addition of barium hydroxide to pH of 9.0.
• the barium hydroxide addition stops enzyme action immediately and causes precipitation of impurities which can be removed from the 5'-nucleotide solution by filtration. This method may result in the removal of small amounts of the desired 5'-nucleotides, particularly purine nucleotides.
• a second method of removing impurities involves the addition to the hydrolysis solution of alcohol to stop enzyme action and precipitate protein and some sodium phosphate. Small amounts of purine nucleotides are also precipitated. The precipitate is filtered off and the filtrate used to separate 5-nuc1eotides.
• the filtrate (after removal of impuri ties) contains nucleotides which may be separated by well-known ion-exchange techniques.
• an enzymic media comprised of water and uncomminuted seed-parts (which has been heat treated in a manner similar to that described herein before) may be utilized to degrade or digest deoxyribonucleic acid to form 5'-deoxyribonucleotides.
• An aqueous enzymic medium capable of digesting ribonucleic acid to form primarily 5'-ribonucleotides comprising a dispersion of discrete rapidly proliferating substantially non-comminuted seed-parts selected from the group consisting of substantially seed-free germinating seed rootlets and substantially seed-free germinating seed stems in water, said mixture having been heated to from between about 70 to about 85 C..for from about 0.5 to about 7 minutes.
• a method for the preparation of an aqueous enzymic medium capable of digesting ribonucleic acid to form primarily 5-ribonucleotides which comprises:

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Citations (3)

• 1964
  • 1964-09-10 FR FR987735A patent/FR1438228A/fr not_active Expired
  • 1964-09-14 BE BE653073D patent/BE653073A/xx unknown
  • 1964-09-15 NL NL6410733A patent/NL6410733A/xx unknown
  • 1964-09-29 DE DE19641470320 patent/DE1470320A1/de active Pending
  • 1964-10-05 GB GB40427/64A patent/GB1026639A/en not_active Expired
• 1965
  • 1965-10-04 US US492875A patent/US3304238A/en not_active Expired - Lifetime

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